Bioluminescence is a phenomenon based on a chemical reaction in vivo, which is called a luciferin (a luminescence substrate)-luciferase (an enzyme that catalyzes the luminescence reaction) reaction. Numerous studies of the identification of luciferins or luciferases and the elucidation of the luminescence mechanism in a molecular level have been performed for a long time in the country and overseas. In bioluminescent marine organisms, Oplophorus gracilirostris luciferase from the deep-sea shrimp is an extracellularly secreted luciferase (Non-Patent Document 1). Oplophorus luciferase is a 106 kDa protein composed of a protein with a molecular weight of 35 kDa and a protein with a molecular weight of 19 kDa. The domain that catalyzes the luminescence is found to be 19 kDa protein. Oplophorus luciferase uses coelenterazine as a luminescence substrate and is classified as a coelenterazine-type luciferase (Patent Document 1, Non-Patent Document 2). Oplophorus luciferase is different from other coelenterazine-type luciferases in broad substrate specificity and uses coelenterazine analogues as a suitable substrate as well as coelenterazine (Non-Patent Document 2). When the gene for the 19 kDa protein is expressed in Escherichia coli (E. coli) at ambient and lower temperatures, the protein is expressed mostly as an insoluble protein (Non-Patent Document 3). When the 19 kDa protein is expressed as a fusion protein to ZZ domain from protein A in a low temperature expression system, the fused protein can be expressed as a soluble protein (Non-Patent Document 4). It is also reported that when the 19 kDa protein was expressed in animal culture cells, the expressed protein was hardly secreted outside of cells (Non-Patent Document 2).
Recently, it is reported that the mutated 19 kDa protein having catalytic activity of luminescence was prepared by mutating the 16 amino acids of the 19 kDa protein and showed a higher luminescence activity than native 19 kDa protein, and was secreted into an extracellular medium (Patent Document 2, Non-Patent Documents 4 and 5). It is also reported that coelenterazine derivatives displayed higher activity than native coelenterazine used as a substrate (Non-Patent Documents 4 and 5).
In the luminescence reaction system using coelenterazine as a substrate, the luminescence reaction of luciferase proceeds only by a substrate and molecular oxygen. For this reason, a coelenterazine-type luciferase gene is used widely as a reporter assay in an animal cultured cell system at present. Renilla luciferase having 311 amino acids is used for a reporter assay inside of cells. For an extracellular reporter assay, the secreted Gaussia luciferase which is a secretory luciferase having 168 amino acids is used. When recombinant Renilla luciferase and Gaussia luciferase are compared in specific activity using coelenterazine as a luminescence substrate, the specific activity of Renilla luciferase is about 1/100 of Gaussia luciferase (Non-Patent Documents 5 and 6). On the other hand, the specific activity of mutated 19 kDa protein having catalytic activity of luminescence is 1/10 as compared to Gaussia luciferase, indicating that the mutated 19 kDa protein is obviously inferior as the gene for a reporter assay as a secreted protein.
In view of the foregoing, it has been desired to develop a reporter gene which is an intracellular and secreted luciferase and has a higher luminescence activity than that of native 19 kDa protein when coelenterazine or even its analogue is used as the substrate.